Report of a new species of sand fly, Phlebotomus (Anaphlebotomus) ajithii n. sp. (Diptera: Psychodidae), from Western Ghats, India

Background Western Ghats is a biodiversity treasure trove with reports of indigenous leishmaniasis cases. Hence, systematic sand fly surveillance was carried out among the tribal population. The present study reports a novel sand fly species, Phlebotomus (Anaphlebotomus) ajithii n. sp. (Diptera: Psychodidae), discovered in the Western Ghats of India. Methods A comprehensive sand fly survey was conducted across the Kollam, Thrissur, Idukki, Kasaragod and Malappuram districts of Kerala, India. The survey spanned both indoor and outdoor habitats using standard collection methods over a 3-year, 3-month period. DNA barcoding of samples was performed targeting mitochondrial cytochrome c oxidase subunit I (COI) gene, and the sequence generated was subjected to phylogenetic analysis. Results Phlebotomus (Anaphlebotomus) ajithii, a new sand fly species, is recorded and described in this communication. The morphological relationship of the new species to other members of the subgenus Anaphlebotomus is discussed. Mitochondrial COI barcode followed by phylogenetic analysis confirmed that specimens of Ph. ajithii belong to the same taxonomic group, while a genetic distance of 11.7% from congeners established it as a distinct species. Conclusions The Western Ghats, known for its rich biodiversity, has lacked systematic entomological surveys focusing on sand flies. This study aims to fill this gap and reports and describes a new species of sand fly. Graphical abstract


Background
Phlebotomine sand flies are classified within the Psychodidae family under the order Diptera, and they are diminutive, hairy insects that feed on blood.These insects are of immense public health importance since they play a role as vectors of leishmaniasis.Leishmaniasis, a neglected tropical disease (NTD), is caused by a flagellate protozoan parasite from the Leishmania genus.It has been endemic in many countries like India, Brazil, Sudan, Bangladesh, Ethiopia and Nepal for centuries [1].
The Western Ghats, also known as the Sahyadri, is a biological treasure trove that boasts a rich biodiversity comprising numerous endemic species of plants, fishes, amphibians, reptiles and mammals [2,3].In India, the Western Ghats expands from Gujarat to Tamil Nadu with an area of 160,000 km 2 and is considered one of 34 biodiversity hotspots worldwide [4].Several tribal populations belonging to different ethnic groups are inhabitants of this zone.Human inhabitation and the practice of agriculture have made several alterations in the ecology and landscape of the Western Ghats [5].In addition, this region is also relevant in terms of different vector-borne diseases, transmitted by insects, ticks, mites, etc. [6][7][8].
Western Ghats is emerging as an endemic belt for visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) in the last few decades, with many new indigenous case records [9].Over the last 2 decades, nearly 50 cases of CL and VL were reported from the tribal population of these zones [10,11].Sand flies are the only proven vector of the Leishmania parasite involved in the transmission of leishmaniasis.The availability of resting and breeding habitats and the many blood meals via various hosts have aided in the abundant survival of sand flies in this region [12].From 1990 till 2022, many new species and country records of sand flies have been documented from the Western Ghats through various entomological survey carried out by a few researchers [13][14][15][16][17][18][19][20][21][22].Later, all these species were combined and included in a review article with a total of the 69 sand fly species recorded in India; almost 50% those species are found in Kerala [23].Hence, considering the rich biodiversity and endemicity of the disease, systematic sand fly surveillance has been carried out across the Western Ghats of Kerala, especially in the tribal belts.A new species of sand fly was identified during this survey, Phlebotomus (Anaphlebotomus) ajithii n. sp.Taxonomic and phylogenetic characterizations of the species are documented in this article.

Study area
The entomological investigation was conducted within the various tribal communities situated in Western Ghats of Kollam, Thrissur, Idukki, Kasaragod and Malappuram districts in Kerala from January 2021-March 2024 (Table 1 and Fig. 1).
These tribal settlements are scattered throughout the mountainous forest expanse of the Western Ghats in southern India.Referred to locally as the Sahyadri, this elevated area in Kerala lies on the periphery of the Deccan Plateau, demarcating it from the coastline strip along the Arabian Ocean.The Ghats boasts diverse vegetation, encompassing grasslands, arid and humid temperate forests, evergreen and semi-evergreen woodlands, scrub jungles and more.Coconut, rubber, pepper, jackfruit and teak plantations within these forests serve as major sources of livelihood for the indigenous tribes.The rich cultural heritage of the region is safeguarded by its intricate topography and abundant rainfall.Moreover, the forests within these ranges are designated as reserve forests and are patrolled by forest rangers appointed by the Kerala Government.
The tribes residing within these mountain ranges now inhabit dwellings constructed of concrete blocks, a measure introduced by the Kerala Government as part of an initiative aimed at the development of the tribal population.Reliant on the forest for their livelihood, these tribes often migrate to the remote reaches of the forest.Consequently, the abandoned houses, left vacant amid the moisture and humidity brought by the rains, create an ideal microhabitat for the breeding of sand flies.Within these settlements, houses are spaced roughly 100-200 m apart, totaling around 50-100 dwellings.Sand fly collection was conducted using standard entomological collection methods such as mechanical aspirators, light and sticky traps both indoors (cattle sheds and human dwellings) and outdoors (rodent burrows, tree holes, termite mounts etc.).On the other hand, resting collections employing mechanical aspirators were carried out in the dawn hours, between 9:00 a.m. and 12:00 noon.Trap collections, on the other hand, were undertaken from 6:00 p.m. (the preceding day) to 6:00 a.m.(the following day), covering the evening and night hours.

Morphological identification
Sand fly specimens were brought to Indian Council of Medical Research-Vector Control Research Centre (ICMR-VCRC), Field Station at Kottayam, and preserved in 70% ethanol.The samples were dissected under  2) [13,24,25].Some specimens (female and male) did not match the characters of the reported species in the keys and published literature.Taxonomic features of these specimens are distinct from the congeners sand fly species (subgenus Anaphlebotomus) such as Phlebotomus (Ana.)colabaensis Young and Chalam, 1927, Ph. (Ana.)stantoni Newstead, 1914, and Ph.(Ana.)hoepplii Tang and Maa, 1945, which have been previously explained [13].The morphometric analysis of the possible new sand fly species was carried out using a Zeiss binocular microscope aided with a micrometer.Every measurement was logged in micrometers (µm).Images of unique identifying features of these specimens were taken using a camera mounted over the same compound microscope.A holotype female and allotype male along with nine paratype female samples were used for morphometric analysis.Terminologies of the attributes for description were adopted from Galati et al. [26].Nomenclature was adopted following the guidelines given by the International Code of Zoological Nomenclature (ICZN) [27].Measurements (µm) of Ph. (Ana.)ajithii n. sp.(holotype female and allotype male) are described below (Table 3).

Molecular identification
The genomic DNA was extracted from both the legs and entire body of individual sand flies using the QIAmp DNeasy Kit (Qiagen, Germany), adhering to the manufacturer's instructions.The sand fly specimen was homogeneously crushed with a mortar and pestle, and final DNA elution was done in 30 μl molecular-grade nuclease-free water.DNA barcoding of both female and male samples was performed targeting mitochondrial cytochrome c oxidase subunit I (COI) gene (~ 720 bp).
The gene was amplified according to the procedure outlined by Kumar et al. [28].Sequencing of the amplicons was done bi-directionally using the same set of primers.The nucleotide sequences generated were deposited in GenBank.

Phylogenetic analyses
For phylogenetic analysis, the sequences were blasted with nucleotide repository (GenBank), and the sequences most similar to resulting sequences were aligned using MEGA 7.0.The sequences aligned with other congeners were analysed for phylogenetic tree construction using neighbor-joining statistical method with Kimura 2.0 parameter and 10,000 bootstraps.The genetic distance and other related parameters were estimated using MEGA 7.0.

Natural infection assessment of Leishmania parasite
Since the possible new species of sand fly belongs to the Phlebotomus genus, assessment for natural infection of Leishmania parasite was carried out.The whole genomic DNA was subjected to real-time detection of Leishmania kinetoplast minicircle DNA (kDNA).The protocol for kDNA detection was followed as described by Castelli et al. [29] using primers LEISH-1 (5ʹ GGC GTT CTG CGA AAA CCG 3ʹ), LEISH-2 (5ʹ AAA ATG GCA TTT TCG GGC C 3ʹ) and TaqMan probe (5ʹ FAM-TGG GTG CAG AAA TCC CGT TCA 3ʹ-BHQ1).
Cibarium: the ventral area bears a few distinct horizontal teeth but no denticles or fore-teeth; pigment patch was absent on the dorsal plate of the cibarium.Cibarium bears about 4-5 varying distinct horizontal teeth.All the teeth are irregularly arranged, tapering to fine points.Pharynx: nearly slender in shape with relatively broad base, armed heavily with a small group of minute spicules, length 189 µm, width 74 µm, pharyngeal armature depth 63 µm.Pharynx is about 2.5 times as long as wide.Wings: length 1786 µm, width 567 µm.Length of principal vein sections: alpha 430 µm, beta 283 µm, gamma 232 µm, delta (R1 overlap) 102 µm, R5 length 1252 µm.Wing index (alpha/beta) 1.51.
Genitalia: Spermathecae: tubular shaped, slightly narrow towards duct end with 13-15 clear distinct segmentations.Length 39 µm, width 16 µm.Apical segment of the spermatheca is not enlarged (short neck).Spermatheca with secretary cells at the distal end and narrow individual spermathecal duct with striations.The length of the individual spermathecal duct is about 15-20 times (585-780 µm) the length of the spermatheca, and the  No SS present on f1 to f10.Four SS on f11, 10 on f12, 10 on f13 and 20 on f14 were observed.Single distal papilla observed on f1 to f3.Papilla absent from f4 to f11.One papilla on f11, three on f12, three on f13 and two on f14 were observed.Cibarium: no visible horizontal teeth, denticles or foreteeth, pigment patch was absent on the dorsal plate of the cibarium.Pharynx: nearly slender in shape with marginally broad base, comparatively lightly armed compared with female specimen, with a small group of minute spicules, length 158 µm, width 44 µm, pharyngeal armature depth 43 µm.Pharynx is about 3.6 times as long as wide.
Genitalia: length of sperm pump is 204 µm, length of aedeagal duct is 175 µm, length of sperm pump + length of aedeagal duct: 378 µm, ratio of length of sperm pump/ length of aedeagal duct is 1.17.Aedeagal duct is much shorter than the length of the spermathecal duct of female specimen.The aedeagal filament has no striation, i.e. it is smooth, straight, with rounded ends and overall slender throughout the duct.Ejaculatory apodeme of the male genitalia; 152 µm long.Gonocoxite of length 203 µm and with a median tuft of 48-50 internal setae.Gonostyle with a length of 150 µm and four thick spines: one apical, two subapical or subterminal and one basal with average length of 108 µm.Complex paramere with two lobes: long upper lobe with length of 148 µm and about 60 strong upward-facing setae, another shorter lobe (ventral process) without any such setae.Parameral sheath or aedeagus is thick distal end with tapering end and is finger-like in structure with a length of 102 µm.Length of epandrial lobes is 226 µm (Fig. 3).

Diagnosis
The characteristics of uniformly erect hairs on abdominal tergites (2-6) found in the specimen are particular to the genus Phlebotomus.Cibarium has spicules, i.e. fine tooth-like structures, but not in a consistent row.Pigment patch is absent.Pharynx is armed with a minute group of teeth in the middle and behind it a few concentric lines.Palps extending further than antenna III (f3).Spermatheca has regular segmentation.Apical segment of the spermatheca is not enlarged, i.e. it has a short neck.Chitinous arch is well developed along with   proximal end.The aedeagal duct was short in length and was smooth and slender in shape.These features are distinctive in holotype female and allotype male Ph.(Ana.)ajithii n. sp.specimens.

Variability
The morphometric characteristics indicated that the holotype and paratype of female and allotype and paratype of males were similar (Table 3).Holotype, allotype and paratype female and male were collected from the same type of habitats but different districts.All specimens of Ph. (Ana.)ajithii n. sp.showed similarities in the taxonomic characteristics.In addition, DNA barcode sequences of the collected specimens from different districts showed variation in four nucleotides and an overall negligible genetic distance (K2P) within the specimens, thus suggesting a single taxonomic group.However, the genetic distance from the other most comparable congeners was 11.7% [Ph.(Ana.)colabaensis: 16.7%; Ph. (Ana.)stantoni: 17.7%] (Fig. 4).

Type materials
Phlebotomus (Ana.)ajithii constitutes about 4.9% (69 specimen) of the total species composition.The type locality of Ph. ajithii n. sp. is cattle sheds and adjacent rooms from Kadaar tribal community of Mullurkara (Thalapilly taluk) (GPS coordinates 10.69"N, 76.25"E, altitude: 103 m above sea level).This new species has also been caught from same habitat in different districts as described in Table 1.Paratype females were obtained from the Kadaar tribe of Mullurkara tribal settlements Thrissur, Kerala, India.Paratype males were collected from Ottamala settlement, taluk-Vellarikund, Panchayat-Panathadi, District-Kasaragod, Kerala, India: (12.48"N, 33"E, altitude: 158 m above sea level).Voucher specimens, comprising holotype female and allotype male, were mounted separately on microscopic glass slides and serially numbered with details about the place and date of collection, habitat type, etc., and were deposited at the museum, ICMR-VCRC), Puducherry-605 006, India.Additionally, paratype female and male were subsequently submitted to the National Museum, Zoological Survey of India, Alipur, Kolkata, India.

Etymology
The new species Ph. (Ana.)ajithii is named after Mr. PM Ajithlal (Technical Officer 'C' [Retired]), ICMR-VCRC, Field Station, Kottayam, Kerala) in recognition of his unwavering commitment and lifelong dedication to the field of public health entomology.

Natural infection assessment of Leishmania parasite
None of the Ph.(Ana.)ajithii samples were found positive for Leishmania parasite.

Discussion and conclusion
The distribution of sand flies from the rain forests of Western Ghats in India was abridged and updated by Lewis [13,25].The present systematic entomological survey was carried out in various tribal villages situated in the Western Ghats region of different districts in Kerala.These areas were selected because of their epidemiological relevance, i.e. case reports of leishmaniasis.These areas provide favourable macro-and microhabitat (rainfall, organic-rich soil and variety of hosts as blood sources, etc.) for the proliferation and abundance of sand flies all year.Additionally, this investigation provides data on the record of a new sand fly species from these tribal settlements.
There are several genera of sand flies, of which genus Phlebotomus Rondani and Berté in Rondani 1840, comprises many species from the Old World [13,24].Subgenera Euphlebotomus and Anaphlebotomus are the only two subgenera in which some species share common female characteristics, exhibiting pharyngeal armature, being armed and having cibarial teeth or spicules.However, females of both subgenera can be distinguished based on the apical segment of the spermatheca [13].Species under the subgenus Euphlebotomus exhibit the peculiar characteristics of apical segments of the spermatheca being differentiated or enlarged compared with the others by a deeper furrow [13,24,30].
The subgenus Anaphlebotomus was one of the smallest groups under the genus Phlebotomus, including a total of only three species from the Oriental region [13,24].Phlebotomus (Ana.)colabaensis, Ph. hoepplii and Ph.stantoni are the species of the subgenus Anaphlebotomus which were recorded from the country [13,24].In 2023, this was revised to five species by Shah et al. in the review article entitled "Faunal richness and checklist of sand flies (Diptera: Psychodidae) in India" with two species records from India, i.e.Phlebotomus (Ana.)chiyankiensis Singh, Phillips Singh and Ipe, 2009, and Ph.(Ana.)palamauensis Singh, Phillips Singh and Ipe, 2007 [23].However, as discussed by Renaux et al. in 2023, the validation of these species cannot be confirmed based only on the drawings and morphological descriptions provided by the authors [31].Furthermore, we also accept the revision of the systematics of the subgenera Anaphlebotomus and reinstated the validity of Phlebotomus maynei, based on examination of its holotype by Renaux et al. [31].In this continuation, a new species record, Ph. (Ana.)ajithii n. sp., is described in this article.
Referring to the standard taxonomic keys, the female specimens of Ph. (Ana.)ajithii n. sp. were found to be similar to females and males of the congener, such as Ph.(Ana.)colabaensis, Ph. stantoni and Phlebotomus shadenae [13,24,31].However, based on some peculiar taxonomic characteristics, those plausible new species specimens were separated.In Ph. (Ana.)colabaensis female, the labrum is about 300 µm long.Hypopharynx has about 19 teeth on each side and maxilla with 11 lateral and 23 ventral teeth.Ascoid in f2 is about 60 µm.Spermatheca is slightly carrot shaped with a small end segment.Spermathecal duct is long, i.e. about four to five times length of the individual spermatheca which joins to the common duct [13,24].In Ph. (Ana.)colabaensis male, the terminalia has four spines on the gonostyle with two being apical and subapical and one basal in position.Sperm pump is 150 µm long.Gonocoxite and gonostyle are about 150 and 230 µm long, respectively.Aedeagus or parameal sheath is pyramidal in shape and sharply pointed [31].In Ph. (Ana.)stantoni female, the labrum is about 230-260 µm long.Hypopharynx has about 16 teeth on each side and maxilla with nine lateral and 18 ventral teeth.Ascoid in f2 is about 80 µm.Spermatheca is fusiform or spindle shaped with about 15-16 segments; the neck is thick and short with a more or less oblong head.The spermathecal duct is slightly longer than the length of the individual spermatheca and is striated.Common duct is very long, i.e. about 1.5 times the length of the spermatheca with thick walls [13,24].In Ph. (Ana.)stantoni male, labrum is about 169 µm long.Gonostyle is 77 µm long, and sperm pump is 143 µm long.Ejaculatory apodeme is about 107 µm long.Gonocoxite is 190 µm long with 43 internal gonocoxal tuft setae.Aedeagus or parameal sheath is 88 µm long [31].For Ph. shadenae female, labrum is 187 µm long.Hypopharynx has about 15 teeth on each side and maxilla with 8 lateral and 15 ventral teeth.Individual spermathecal duct is 69 µm long and has spermatheca with > 15 rings along with the presence of a sessile head carried by a broad process [31].In Ph. (Ana.)shadenae male, labrum is about 167 µm long.Gonostyle is 84 µm long with four thick spines, one of which is terminal and one subterminal, and two are basal in position.Sperm pump is 139 µm long.Ejaculatory apodeme is about 107 µm long.Gonocoxite is 207 µm long with 56 internal gonocoxal tuft setae.Aedeagus or parameal sheath is 88 µm long and is finger-like in shape.Aedeagal ducts are straight, smooth and overall tapering and slender with rounded ends [31].However, Ph. (Ana.)ajithii n. sp.female has about 270-µm-long labrum.The hypopharynx has about 18 teeth on each side, and the maxilla has 10 lateral and 17 ventral teeth.Ascoid present on f2 is about 68 µm.The apical segment of the spermatheca is not enlarged (short neck), which is one of the key features that differs between the two subgenera, i.e.Euphlebotomus and Anaphlebotomus [13,24,25].Spermatheca is tubular and slightly narrow towards the duct end with about 13-15 segmentations.Spermathecal ducts are narrow with striations.The individual spermathecal duct is long, about 15-20 times (585-780 µm) the length of the spermatheca, and is highly coiled.It has a short common spermathecal duct of 54 µm length (Fig. 2).In the Phlebotomus (Ana.)ajithii n. sp.male, labrum is about 183 µm long.Ascoid length on f2 is 51 µm.Gonostyle is 150 µm long with four thick spines, one apical, two subapical or subterminal and one basal in position, with average length of 108 µm.Sperm pump is 204 µm long.Ejaculatory apodeme is about 152 µm long.Gonocoxite is 203 µm long with 48-50 internal gonocoxal tuft setae.Aedeagus or parameal sheath is 102 µm long and has a thick distal end with tapering end.Aedeagal ducts have no striation, i.e. they are its smooth and straight with rounded ends and are overall slender throughout the duct.
In addition, molecular taxonomy by DNA barcoding followed by phylogenetic analysis also confirmed the association within the specimens of Ph. (Ana.)ajithii n. sp. with a very minimal genetic distance and four nucleotide variations.However, the overall genetic distance (GD) is 11.7% with the congener species.Phlebotomus (Ana.)colabaensis has 16.7% and Ph.(Ana.)stantoni has 17.7% GD with Ph. ajithii.The population genetic parameters analysed using MEGA 7.0 software also confirmed a very high genetic diversity (H ST = 0.969) and trifling gene flow (N m = 0.002).Hence, based on these taxonomic differences and molecular analysis, Ph. (Ana.)ajithii n. sp. is divergent from the other already reported and described species under the subgenus Anaphlebotomus.This species was mainly collected from indoor human dwellings since species of the genus Phlebotomus are mainly incriminated in the transmission of diseases [32][33][34][35][36], thus, infection assessment was carried out.However, none of the specimens were positive for the Leishmania parasite in qPCR targeting kDNA.In conclusion, the Western Ghats is an important biodiversity hotspot with a few database on entomological surveys of sand flies.The current study tried to fill this void and also report a new sand fly species.

Fig. 1
Fig. 1 Sand fly specimen collection area in Thiruvananthapuram, Kollam, Thrissur, Idukki, Kasaragod and Malappuram districts of Kerala, India, along with species composition distinct horizontal teeth in the cibarium region of the head.Terminalia is short or medium sized.Paramere with or without short ventral process.Gonostyle with four long spines and coxite without basal process.These taxonomic characters confirm the addition of the species into subgenus Anaphlebotomus of genus Phlebotomus.Cibarium had a few distinct horizontal teeth in female but was rudimentary in male specimens.Maxillary teeth range between 15-18 on the internal side and 9-11 on the external side in the mouth parts of females.Ascoid on second antennal segment (f2) almost crossed the joint between f2 and f3 in both sexes.Spermatheca was tubular in shape with 13-15 clear distinct segmentations.The individual spermathecal duct was 15-20 times (585-780 µm) the length of individual spermatheca and was highly coiled.In males, gonostyle had four thick spines, one apical, two subapical or subterminal and one basal.Paramere with one ventral process and aedeagus with thick distal area and tapered at the

Table 1
Details of entomological survey study area

Table 2
Species composition of phlebotomine sand flies collected and identified in the present study

Table 3
(continued) N number of specimens, Max maximum, Min minimum, SD standard deviation, R radius, '-' not applicable